GSK3 polypeptides

ABSTRACT

The invention provides truncated GSK3 polypeptides capable of crystallization, including GSK3α and GSK3β polypeptides, and use of these polypeptides to identify and optimize GSK3 inhibitors. Also provided are GSK3 polypeptides having at least one substituted amino acid that differs from wild-type GSK3, wherein the substituted amino acid is incapable of being phosphorylated. The invention finds use in providing methods of identifying and optimizing compounds useful for treating diseases mediated by GSK3 activity, including Alzheimer&#39;s disease, type 2 diabetes, and inflammation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/733,816 filed Dec. 10, 2003, which is a divisionalapplication of U.S. patent application Ser. No. 10/211,412 filed Jul.31, 2002, which is a divisional application of U.S. patent applicationSer. No. 09/916,109 filed Jul. 25, 2001, which claims the benefit ofU.S. Provisional Patent Application No. 60/221,242 filed Jul. 27, 2000,where this provisional application is incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention provides materials and methods relating to identificationand optimization of selective inhibitors of glycogen synthase kinase 3(GSK3), and also relates to methods of treating a condition mediated byGSK3 activity. Such conditions include Alzheimer's disease, type 2diabetes, and inflammation.

2. Description of the Related Art

Glycogen synthase kinase 3 (GSK3) is a proline-directed serine/threoninekinase originally identified as an activity that phosphorylates glycogensynthase as described in Woodgett, Trends Biochem Sci. 16:177-181(1991). The role in glucose metabolism has been elaborated recently inSummers et al., J. Biol. Chem. 274:17934-17940 (1999). GSK3 consists oftwo isoforms, α and β, and is constitutively active in resting cells,inhibiting glycogen synthase by direct phosphorylation. Upon insulinactivation, GSK3 is inactivated, thereby allowing the activation ofglycogen synthase and possibly other insulin-dependent events. GSK3 isinactivated by other growth factors or hormones that, like insulin,signal through receptor tyrosine kinases. Examples of such signalingmolecules include IGF-1 and EGF as described in Saito et al., Biochem.J. 303:27-31 (1994), Welsh et al., Biochem. J. 294:625-629 (1993), andCross et al., Biochem. J. 303:21-26 (1994). GSK3 has been shown tophosphorylate β-catenin as described in Peifer et al., Develop. Biol.166:543-56 (1994). Other activities of GSK3 in a biological contextinclude GSK3's ability to phosphorylate tau protein in vitro asdescribed in Mandelkow and Mandelkow, Trends in Biochem. Sci. 18:480-83(1993), Mulot et al., Febs Lett 349: 359-64 (1994), and Lovestone etal., Curr. Biol. 4:1077-86 (1995), and in tissue culture cells asdescribed in Latimer et al., Febs Lett 365:42-6 (1995). Selectiveinhibition of GSK3/may be useful to treat or inhibit disorders mediatedby GSK3 activity.

There is a need in the art for compositions and molecules that bind toor interact with GSK3, thereby mediating GSK3 activity. The inventionmeets this need by providing crystallizable GSK3 polypeptides useful fordesign and optimization of GSK3 inhibitors.

BRIEF SUMMARY OF THE INVENTION

The invention provides GSK3β molecules with N— and C-terminaltruncations, wherein the molecules are capable of crystallization.

The invention further provides GSK3β molecules truncated at amino acidR³⁴⁴, R³⁵⁴, T³⁶⁴ , A³⁷⁴ , and I³⁸⁴.

The invention provides a polypeptide consisting essentially of SEQ IDNO:2 or SEQ ID NO:3, polynucleotides encoding these polypeptides, andvectors comprising these polynucleotides.

The invention still further provides GSK3β molecules wherein translationof the molecule begins at G³⁴, T³⁹, P⁴⁴, D⁴⁹ or V⁵⁴.

The invention also provides GSK3α molecules with N— and C-terminaltruncations, wherein the molecules are capable of crystallization.

The invention further provides a GSK3α molecule wherein translation ofthe molecule begins at S⁹⁷ and ends at S⁴⁴⁷, polynucleotides encodingthis polypeptide, and vectors comprising these polynucleotides.

The invention further provides a method of identifying a GSK3polypeptide capable of crystallization, comprising: (a) providing atruncated GSK3 polypeptide; (b) testing the polypeptide for formation ofcrystals.

The invention also provides GSK3 polypeptides capable of interactingwith inhibitors of GSK3.

The invention further provides a method of identifying an enzymaticallyactive GSK3 polypeptide, comprising: (a) providing a truncated GSK3polypeptide; (b) contacting the polypeptide with a substrate of GSK3;and (c) measuring the kinase activity of the polypeptide aftercontacting the polypeptide with the substrate, wherein the polypeptideis active if it shows >0.01×the activity of the full-length enzyme andpreferably >0.1×the activity of the full-length enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

FIG. 1 provides the polypeptide sequence of human GSK3β (SEQ ID NO:1).

FIG. 2 provides the polypeptide sequence of truncated GSK3β polypeptide557 (SEQ ID NO:2). The first ten amino acids represent a Glu-tag,followed by a Gly linker before Met at position 1.

FIG. 3 provides the polypeptide sequence of truncated GSK3β polypeptide580 (SEQ ID NO:3). The first ten amino acids represent a Glu-tag,followed by a Gly linker before Gly at position 34.

FIG. 4 provides the polypeptide sequence of human GSK3α (SEQ ID NO:4).

FIG. 5 provides the polypeptide sequence of human GSK3α truncated atposition 447 (SEQ ID NO:5).

FIG. 6 provides the polypeptide sequence of human GSK3α truncated atposition 97 (SEQ ID NO:6).

FIG. 7 provides the polypeptide sequence of human GSK3α from position 97to position 447 (SEQ ID NO:7).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides materials and methods for identifying andoptimizing inhibitors of GSK3, including GSK3α and GSK3β. The providedmaterials include C— and N-terminal truncated GSK3β molecules that arecapable of crystallization and may, but need not, retain GSK3 kinaseactivity, preferably more than 0.01×the activity of the full-lengthenzyme and more preferably more than 0.1×the activity of the full-lengthenzyme. There is a need in the art for such inhibitors, in view of therole of GSK3 in a variety of diseases and conditions, includingAlzheimer's disease, type 2 diabetes and inflammation. Such inhibitorscan be identified, and identified inhibitors can be optimized, using thecrystallizable GSK3 polypeptides of the invention.

The invention provides a variety of GSK3β polypeptides that differ fromthe native polypeptide at the C— and/or N-terminus. The amino acidsequence of GSK3β is shown in FIG. 1 (SEQ ID NO:1). Included within thescope of the invention are any and all truncations of GSK3β polypeptidewherein the truncated polypeptide is capable of crystallization and may,but need not, retain kinase activity as measured using the kinase assaysdescribed herein. Persons of skill in the art will realize that limitedmutation of the protein, or certain post-translational modifications,might be sufficient to inactivate the kinase yet retain the essential 3Dstructure. Such inactive but structurally related molecules would alsobe useful for the design and optimization of inhibitors. Kinase assaysare disclosed in U.S. Pat. Nos. 6,057,117 and 6,057,286, which areincorporated herein by reference. The percent activity that is retained,if any, is not crucial. Methods of assaying activity in the presence andabsence of an inhibitor are described herein.

The invention provides numerous truncated GSK3β polypeptides that meetthese criteria. A preferred polypeptide is designated BV557 in which theC-terminal amino acid is R³⁸⁴. This molecule has been successfullycrystallized. Additional active polypeptides include those withtruncations at amino acid R³⁴⁴, R³⁵⁴, A³⁷⁴, and I³⁸⁴.

The invention also provides truncated GSK3α polypeptides, including aGSK3α polypeptide beginning at S⁹⁷ and ending at S⁴⁴⁷.

Additional truncated GSK3 polypeptide include those beginning with anN-terminal amino acid that differs from that of the native protein inthat 1 or more amino acids are deleted from the N-terminus. PreferredN-terminal truncations include GSK3β molecules wherein translation ofthe molecule begins at G³⁴, T³⁹, P⁴⁴, D⁴⁹ or V⁵⁴. An example is BV580(amino acids 34 to 384) which has been crystallized.

The invention is not limited to these disclosed truncated molecules.Using the methods and assays described herein, one of skill canconstruct additional truncated molecules, such as those having 36-76amino acids deleted from the C-terminus, and/or 35-54 amino acidsdeleted from the N-terminus. Such deletions can occur individually, or apolypeptide can have both an N-terminal deletion and a C-terminaldeletion. It is preferable but not necessary that the kinase domainremain relatively intact as reflected by the detection of enzymaticactivity, such as by using the assays described herein. It is alsodesirable, although not essential, that the enzymatic activity becapable of inhibition by a known GSK3 inhibitor, such as lithium. Atruncated molecule meeting these criteria will be suitable for testingGSK3 inhibitors as potential therapeutic agents, and for optimizing GSK3inhibitors.

A truncated GSK3β polypeptide of the invention can consist of betweenabout 250 and 419 contiguous amino acids of SEQ ID NO:1; preferablybetween about 278 and 419 contiguous amino acids of SEQ ID NO: 1; morepreferably between about 285 and 384 contiguous amino acids of SEQ IDNO:1; and most preferably between about 351 and 384 contiguous aminoacids of SEQ ID NO:1. Preferred truncated GSK3β polypeptides includethose beginning at amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 of SEQ ID NO:1,and ending at amino acid 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418 or419 of SEQ ID NO:1. The polypeptide can begin with any one of the listedbeginning amino acids and end with any one of the ending amino acids.Exemplary and non-limiting embodiments begin at amino acid 34, 39, 44 or54 and end at amino acid 420. Other particularly preferred embodimentsbegin at about amino acid I and end at amino acid 340, 344, 354, 374,384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, or 420.

The truncated GSK3α polypeptide of the invention can consist of betweenabout 182 and 482 contiguous amino acids of SEQ ID NO: 4, preferablybetween about 182 and 386 contiguous amino acids of SEQ ID NO:4, morepreferably between about 182 and 351 contiguous amino acids of SEQ IDNO:4, and most preferably from about S⁹⁷ to S⁴⁴⁷ of SEQ ID NO:4.

The truncated GSK3 polypeptides can be prepared by any method known inthe art. One method involves expression of a suitably preparedpolynucleotide encoding a polypeptide having the desired truncation. Forexample, a preferred polypeptide of the invention, BV557, was preparedby creating a construct encoding GSK3β starting at M¹ and ending atI³⁸⁴, as described in the Examples. Briefly, insect cells weretransfected with baculovirus vector (designatedpBlueBac4.5.Glu.GSK3B.DC.1384#28), which encodes BU557, and the proteinwas extracted from the lysed cells. The protein was purified by affinitychromatography using an anti glu-tag monoclonal antibody immobilized ona Sepharose column. Activity of the purified protein was assayed usingthe in vitro kinase assay described in U.S. Pat. No. 6,057,286.

The Examples herein describe the production of BV557, BV580, and othertruncated GSK3 polypeptides by expression of vectors encoding thepolypeptides, followed by isolation and purification of thepolypeptides. The polypeptide can also be produced by enzymatic cleavageof a native GSK3 protein, using methods known in the art. Other suitablemethods include expression of a polynucleotide encoding a truncatedpolypeptide in a variety of cell types, including mammalian, bacterial,or yeast cells. However, the preferred cell for expression of thepolypeptide is an insect cell, preferably a baculovirus-infectableinsect cell, such as a Sf9 cell.

The invention also provides unphosphorylated forms of GSK3 wherein theATP binding site is identical to that of the wild-type protein. Suchforms include Y216 non-phosphorylated GSK3β and Y279 non-phosphorylatedGSK3α. Other forms include constructs with at least one amino acidchange that prevents phosphorylation, such as GSK3β in which Y216 ischanged to F216, and GSK3α in which Y279 is changed to F279. These formsare suitable for inhibitor binding assays to identify inhibitors ofGSK3. The invention provides a GSK3β molecule in which position 216 isnot phosphorylated. We have demonstrated that a GSK3β peptide with Y216mutated to F216 crystalized and exhibits a structure in which theATP-binding site is not substantially different from the un-mutatedpeptide.

Additional single and multiple amino acid changes include S⁹ to A⁹ inGSK3β and S²¹ to A in GSK3α.

These changes in phosphorylation, or ability to be phosphorylated, areoptionally incorporated into the truncated forms of GSK3α and GSK3βdisclosed herein.

The invention therefore provides GSK3 molecules suitable for design andoptimization of inhibitors of GSK3 as pharmaceutical agents.

The GSK3 constructs of the invention are capable of crystallization. Inpurified form the constructs bind to inhibitors in a manner that iscomparable to inhibitor binding to the native GSK3 polypeptide, due tothe retention of the correct folding conformation at the inhibitorbinding site. Potential to crystallize is measured using a variety ofassays including specific activity, aggregation, microheterogeneity.(See, for example, Table 1). These parameters are indicative of thepurity of the preparation and of the solubility of the construct. Thespecific activity is also a preferred assay for detecting binding of aninhibitor to the correct binding site of the GSK3 construct. Anothersuitable method is fluorescence polarization. Briefly, a putativeinhibitor, with an attached fluorophore, tumbles freely in solution.Thus when the fluorophore is excited by polarized light, the emittedlight which is produced after a finite delay now has random polarity andthe emitted light is no longer polarized. In the presence of a GSK3construct with an intact inhibitor binding site, the tumbling rate isslowed sufficiently to ensure that, even though the light emission isdelayed with respect to the excitation, the fluorophore has only movedvery slightly. Thus, the excited light maintains polarization. Ameasurement of fluorescence polarization therefore indicates whether ornot the GSK3 construct is suitable for identifying and optimizing aninhibitor. The fluorophore can be attached to a compound such asstaurosporine (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.). GSK3constructs may not retain kinase activity, but their inhibitor bindingcan still be assessed using fluorescence polarization assays.

The term “truncated glycogen synthase kinase 3” or “truncated GSK3” asused herein refers to GSK3α or GSK3β. GSK3 is a protein originallyidentified by its phosphorylation of glycogen synthase as described inWoodgett et al, Trends Biochem Sci, 16:177-181 (1991). Synonyms of GSK3are tau protein kinase I (TPK I), FA kinase and kinase FA. Mammalianforms of GSK3 have been cloned as described in Woodgett, EMBO J.9(8):2431-2438 (1990). Inhibitors of truncated GSK3 polypeptides can beinhibitors of any of the known forms of GSK3, including either GSK3α orGSK3β or both. Truncated polypeptides of the invention possess one ormore of the bioactivities of the GSK3 protein, including kinaseactivities such as polymerizing tau protein, or phosphorylating glycogensynthase, for example. Thus, truncated GSK3 polypeptides useful fordesigning and optimizing inhibitors of GSK3 can have sequence identityof at least 40%, preferably 50%, preferably 60%, preferably 70%, morepreferably 80%, and most preferably 90% to the amino acid sequence ofthe native protein, wherever derived, from human or nonhuman sources.The polynucleotides encoding a GSK3 polypeptide can have 60%, preferably70%, more preferably 80%, more preferably 90% and most preferably 95%sequence identity to a native polynucleotide sequence of GSK3. Alsoincluded, therefore, are alleles and variants of the nativepolynucleotide sequence such that the polynucleotide encodes an aminoacid sequence with substitutions, deletions, or insertions, as comparedto the native sequence.

The term “peptide substrate” refers to a peptide or a polypeptide or asynthetic peptide derivative that can be phosphorylated by GSK3 activityin the presence of an appropriate amount of ATP or a phosphate donor.Detection of the phosphorylated substrate is generally accomplished bythe addition of a labeled phosphate that can be detected by some meanscommon in the art of labeling, such as radiolabeled phosphate. Thepeptide substrate may be a peptide that resides in a molecule as a partof a larger polypeptide, or may be an isolated peptide designed forphosphorylation by GSK3.

As disclosed in U.S. Pat. Nos. 6,057,117 and 6,057,286, in vitro methodsof assaying GSK3 activity include constructing peptide substrates. Thepeptide substrate can be any peptide substrate phosphorylatable by GSK3,and may be a peptide substrate including the formula: anchorligand-(X)_(n)SXXXS(X)_(m) (SEQ ID NO:8) (wherein X is any amino acid, nis any integer, m is any integer, and preferably n+m+5<20, i.e. n+m<15)prephosphorylated at the C terminal serine. The assay is performed bycontacting the prephosphorylated substrate with truncated GSK3polypeptide in the presence of radiolabeled γphosphate-ATP, a substrateanchor, and optionally a candidate inhibitor. The in vitro method ofidentifying an inhibitor of GSK3 kinase activity includes contacting apeptide substrate coupled to an anchor ligand with truncated GSK3polypeptide in the presence of radiolabeled γphosphate-ATP, a substrateanchor, and candidate inhibitor, measuring an incorporation ofradiolabel into the peptide substrate, then, in a separate assay vesselcontacting a peptide substrate coupled to an anchor ligand withtruncated GSK3 in the presence of radiolabeled γphosphate-ATP, and asubstrate anchor, and measuring incorporation of radiolabel into saidpeptide substrate; ultimately an inhibitor of truncated GSK3 kinaseactivity is identified by a reduction of label incorporation in theassay with the candidate inhibitor as compared to the assay without thecandidate inhibitor.

To conduct the in vitro kinase assay of the invention using microwells,scintillant may be present by pre-coating the wells with a scintillantmaterial, or by adding it later following a wash step, as described inExample 4. The scintillant can be obtained from Packard, Meridian, Conn.Wells coated with scintillant are then in addition coated withstreptavidin as a substrate anchor, where biotin is the anchor ligand onthe peptide. Alternatively, the streptavidin can be present on agarosebeads containing scintillant or may be coated on an otherwise untreatedplate to which scintillant is added subsequently. In any event, thestreptavidin in the wells binds the biotin that contacts it. Followingan assay using radiolabeled ATP, the radiolabel incorporated into thephosphorylated substrate that has been conjugated to the biotin willcause the scintillant to emit light. Where the streptavidin is attachedto agarose beads containing scintillant, binding a biotin-conjugatedradiolabeled peptide substrate will cause the beads to scintillate. Inboth the case of the wells lined with the scintillant, and the agarosebeads containing scintillant, a reduction in scintillation as comparedto a control amount of scintillation measured under non-inhibitoryconditions, indicates the presence of a functional inhibitor of GSK3activity. If the peptide has been phosphorylated by GSK3 with³²P-labeled or ³³P-labeled phosphate, radioactive decay will cause thescintillant present in a microwell or mixed in agarose beads that arepresent in the reaction mixture to emit light and the measure of theamount of light emitted will be a measure of the activity of GSK3 in theassay. Low activity of GSK3 observed in the presence of a candidateinhibitor, as compared to the activity of GSK3 in the absence of theinhibitor, may indicate that the inhibitor is functional and can inhibitGSK3 kinase activity. In any case, an excess of streptavidin overpeptide should be loaded into each well or should be affixed to theagarose beads.

GSK3 inhibitory activity can be measured using a cell-free assay asdisclosed in publication WO 99/65897, and as described in Example 4herein. Activity can also be measured using a cell-based assay. Briefly,a cell line, such as a Cos cell line, is transfected with Tau and with aGSK3 polypeptide. The phosphorylation of Tau at a specific site ismonitored using a monoclonal antibody, as phosphorylation at that siteis dependent on GSK3 activity.

Exemplary polypeptides of the invention include the following truncatedpolypeptides with reference to SEQ ID NO:1:

-   -   GSK3β truncated at R³⁴⁴    -   GSK3β truncated at R³⁵⁴    -   GSK3β truncated at T³⁶⁴    -   GSK3β truncated at A³⁷⁴    -   GSK3β truncated at I³⁸⁴    -   GSK3β beginning at G³⁴    -   GSK3β beginning at T³⁹    -   GSK3β beginning at P⁴⁴    -   GSK3β beginning at D⁴⁹    -   GSK3β beginning at V⁵⁴

The above truncations can be combined, providing a GSK3β polypeptidebeginning at any of G³⁴, T³⁹, P⁴⁴, D⁴⁹, or V⁵⁴, and ending at any ofR³⁴⁴, R³⁵⁴, T³⁶⁴, A³⁷⁴, or I³⁸⁴.

Other exemplary polypeptides of the invention include the followingtruncated polypeptides with reference to SEQ ID NO:4:

GSK3α truncated at S⁴⁴⁷.

GSK3α beginning at S⁹⁷.

GSK3α beginning at S⁹⁷ and truncated at S⁴⁴⁷.

A truncated GSK3 polypeptide of the invention can be selected on thebasis of one or more parameters. A polypeptide will preferablycrystallize in a form that is similar to that of native GSK3, withcorrect folding at and around the inhibitor binding site.Crystallization can be performed using a Crystal Screen Kit (HamptonResearch, Laguna Niguel, Calif.), or methods described by Jancarik, J.et al., J. Appl. Cryst. 24:409-411, 1991. The potential of a polypeptideto form crystals can be evaluated on the basis of specific activity,purity, homogeneity, mass spectrometry, aggregation, and dynamic lightscattering. A preferred truncated polypeptide will meet the followingparameters: purity of at least 90%; less than 100% aggregation at 4° C.at two weeks; and less than 50% heterogeneity (50% or greater of thedesired form). A most preferred truncated polypeptide will have a purityof at least 98%, no aggregation at 4° C. at two weeks; and less than 5%heterogeneity (unphosphorylated form). Such parameters indicate that thepolypeptide preparation is likely to crystallize, making it suitable fordiscovering and optimizing GSK3 inhibitors.

A prerequisite for crystallization is to obtain a sufficientlyconcentrated stock of protein. Not all GSK3 constructs will remainsoluble at the required concentration. A preferred concentration is >1mg/ml, more preferred is >5 mg/ml, and most preferred is >10 mg/ml.

The polypeptides disclosed herein as 557 (SEQ ID NO:2), 580 (SEQ IDNO:3), 458, and 524 meet the criteria described above (see Example 3).Polypeptide 458 consists of amino acids 1-420 of SEQ ID NO:1 plus thefollowing addition at the N-terminus: EFMPTEAMAAPKRVI (SEQ ID NO:8).Polypeptide 524 consists of amino acids 1-420 of SEQ ID NO:1 plus thefollowing addition at the N-terminus: EYMPMEGGG (SEQ ID NO:9). Othermodified or truncated GSK3 polypeptides can be prepared and tested asdescribed herein.

EXAMPLES

The following examples are exemplary only, and are not intended to limitthe invention.

Example 1 Preparation and Purification of GSK3β Construct 557

Lysis and Extraction. Insect cell slurry from Sf9 cells (about 10 g)from a 1 liter flask growth was combined with 30 ml of lysate buffer: 20mM Tris, pH 8.0/80 mM NaCl/1 mM MgCl₂/1 mM Arsenate/1 mM Tungstenate/1mM PMSF/0.5 mg Leupeptin/0.2 mg Aprotinin. Cells were lysed using aDounce homogenizer. Improved extraction of the protein was accomplishedby the addition of 5% glycerol and 0.2% octylglucoside. The mixture wasallowed to stir, on ice, for 30 minutes. The total lysate wascentrifuged at 39000×g for 25 minutes at 4° C. The resulting supernatantcontained the extracted GSK3-β #557.

Ion Exchange Chromatography. The following materials and conditions wereused: The resin was Fractogel EMD SO₃— (M); the column diameter was 1.6cm and the column volume was 10 ml. The column was run at a flowrate of90 cm/hour using equilibration buffer of 20 mM Na Phosphate/5% Glycerol,pH 7.5. Chromatography was carried out at 4° C.

The lysate supernatant was diluted 1:1 with S-fractogel equilibrationbuffer, and loaded onto the equilibrated column. The column was washedwith a total of 14 column volumes of equilibration buffer. The GSK3-βwas eluted with a linear salt gradient, over 20 column volumes, toequilibration buffer plus 1 M NaCl. 3 ml/fraction was collected duringgradient elution. The pool was made based on SDS-PAGE and Western blotresults of the fractions collected. Fractions 13-24 were pooled.

Affinity Chromatography was performed using the following materials andprocedures: The resin was anti glu-tag monoclonal antibody immobilizedonto Protein G Sepharose, and the equilibration buffer was PBS/0.3MNaCl/0.2% octylglucoside/10% Glycerol. The column diameter was 1.6 cmand the column volume was 13 ml. The flow rate was 30 cm/hour duringload and wash, and 15 cm/hour during elution.

The S-Fractogel pool was loaded at 30 cm/hour onto equilibrated column.The column was washed down to absorbance baseline with approximately 6column volumes of equilibration buffer, and GSK3β was eluted with 50 mlof equilibration buffer containing 2 mg of elution peptide (EYMPTD). Theflow rate during elution was lowered to 15 cm/hour. 2 ml/fractions werecollected during the elution. Based on SDS-PAGE results, elutionfractions 6-17 were pooled with a total volume of 24 ml.

Final Yield. The affinity column pool, at a concentration of 0.17 mg/ml,contained 4.1 mg of GSK3β #557. This translates to a final yield of 4.1mg purified 557/liter of growth. Purity, after this 2 columnpurification, was estimated at >95% by visual inspection of SDS-PAGEresults.

Example 2 Preparation and Purification of GSK3β Construct 580

Extraction. SF9 cell paste from a 10 L fermentation was washed with 100mL PBS (10 mM NaPi, pH 7.5, 150 mM NaCl) and then resuspended with 300mL of Buffer H (20 mM Tris, pH 7.5, 1 mM Tungstate, 1 mM Arsenate, 5 mMDTT, 10 μg/mL Leupeptin, 1 μg/mL pepstatin A, 10% glycerol, 0.35% Octylglucoside, 1 mM Mg²⁺). Cells were homogenized in a 100-mL DounceHomogenizer (20 strokes with pestle B). The combined homogenate wascentrifuged in a Ti45 rotor at 40,000 rpm for 35 minutes to remove celldebris and nuclei. The supernatant from the centrifugation werecarefully decanted and filtered through 0.45μ filter.

S-Fractogel. 100 mL S-Fractogel (EM Science, Cat #18882) was packed intoa 3.2 cm×12.5 cm column and equilibrated with >1 L of buffer A (20 mMTris, pH 7.5, 10% glycerol). The filtrate from the previous step wasloaded at 15 mL/min onto the column. The column was washed with 1 L ofbuffer A and then eluted with a linear gradient from 0 to 1 M NaCl inbuffer A over 20 column volumes. The eluant was fractionated into 20 mLeach. Fractions containing GSK3 were detected by Western Blot usinganti-GSK antibody (Santa Cruz Biotech, Cat #SC-7291). The Western-Blotpositive fractions were pooled and mixed with equal volume of buffer M(20 mM Tris, pH 7.5, 10% glycerol, 3.1 M NaCl) and filtered through a0.45μ filter. The filtrate was saved for Phenyl-650 M chromatography.

Phenyl-650 M. 37.5 mL Phenyl-650 M (Tosohass, Cat #014943) was packedinto a 2.2×10 cm column and equilibrated with 500 mL of buffer C (20 mMTris, pH 7.5, 10% glycerol, 1.6 M NaCl). Filtrate from S-fractogel stepwas loaded onto the column at 7.5 mL/min. After the loading wascompleted, the column was washed with 6.5 cv buffer C and eluted with alinear gradient from 0% to 100% Buffer D (20 mM Tris, pH 7.5, 10%glycerol) over 20 column volumes. Fractions were collected at 15 mL eachand GSK containing fractions were detected by Western Blot usinganti-GSK antibody. The Western positive fractions were pooled and loadedonto a Glu-tag antibody affinity column.

Glu-tag antibody Affinity Chromatography. Use of a Glu-tag is describedin Rubinfeld et al., Cell 65:1033-1042, 1991, and a hybridoma expressinganti-Glu-tag antibody is described in Grussenmyer et al., PNAS82:7952-7954 (1985). 50 mg of the Glu-tag antibody was immobilized onto25 mL of Affi-Gel 10 (BioRAD, Cat #153-6046) and packed into a 2.2×6.5cm column. The column was equilibrated with 200 mL of buffer E (20 mMTris, pH 7.5, 10% glycerol, 0.3 M NaCl, 0.2% Octylglucoside) and thefraction pool from the Phenyl-650 M step was loaded at 1.0 mL/min. Afterthe loading was completed, the column was washed with 100 mL of buffer Eand then eluted with 60 mL Glu-tag peptide (100 μg/mL) in Buffer E andfractionated into 5 mL each. GSK containing fractions were detected withSDS-PAGE and Coomassie Blue staining. These fractions were pooled andconcentrated to approximately 6 mg/mL in an Amicon concentrator using a10 k MWCO YM10 membrane. The concentrated material was then ready forcrystallization.

Example 3 Activity of Truncated GSK3β Polypeptides

A reaction mixture was prepared containing 5.9 μM prephosphorylatedSGSG-linked CREB peptide (Wang et al., Anal. Biochem., 220.397-402(1994))μ in reaction buffer (5 mM Tris, pH 7.5, 5mM DTT; 1 mM MgCl2,0.01% BSA) containing the desired amount of truncated GSK3 polypeptide.ATP was added (specific activity 5.3 Ci/mmol) to 25 μM finalconcentration and the mixture was incubated for 20 min. at roomtemperature. The reaction was stopped by transferring 30 μl onto a P81filter disc (Whatman). The disc was washed four times in 150 ml of 75 mMH₃PO₄ for 5 minute each. The filter was air dried and counted under 5 mlscintillation fluid. The specific activity was counted by determiningthe ratio of counts (in cpm) by the mass of GSK3 in the reaction (inμg).

The specific activity for construct 557 was 4.3×10⁷ cpm/μg; forconstruct 458, 2.8×10⁷ cpm/μg; and for construct 524, 2.2×10⁷ cpm/μg.TABLE 1 Mean Specific Concen- Activity tration Aggregation AggregationHeterogeneity Construct Purity cpm/μg N mg/ml at 4 Degrees at Rt %458 >98% 2.8 × 10⁷ 35 11.5 11% @ > 2 overnight 10-20% weeksunphosphorylated 557 >98% 4.3 × 10⁷  7 12.7 none @ > 2 overnight  5%weeks unphosphorylated 524 >98% 2.2 × 10⁷ 24 10 ND <5% unphosphorylatedN = number of assays used to determine “mean specific activity.”

Example 4 Screening for GSK3 Inhibitory Activity Using a Cell-Free Assay

Compounds to be tested as GSK3 inhibitors are dissolved in DMSO, thentested for inhibition of human GSK3β. Expression of GSK3β is described,for example, in Hughes et al., Eur. J. Biochem., 203:305-11 (1992),which is incorporated herein by reference. An aliquot of 300 μl ofsubstrate buffer (30 mM tris-HCl, 10 mM MgCl₂, 2 mM DTT, 3 μg/ml GSK3β)and 0.5 μM biotinylated prephosphorylated SGSG-linked CREB peptide(Chiron Technologies PTY Ltd., Clayton, Australia) is dispensed intowells of a 96 well polypropylene microtiter plate. 3.5 μl/well of DMSOcontaining varying concentrations of each compound to be assayed orstaurosporine (a known kinase inhibitor used as a positive control, or anegative control) (i.e., DMSO only), is added and mixed thoroughly. Thereactions is then initiated by adding 50 μl/well of 1 μM unlabeled ATPand 1-2×10⁷ cpm γ³³P-labeled ATP, and the reaction is allowed to proceedfor about three hours at room temperature.

While the reaction is proceeding, streptavidin-coated Labsystems“Combiplate 8” capture plates (Labsystems, Helsinki, Finland) areblocked by incubating them with 300 μl/well of PBS containing 1% bovineserum albumin for at least one hour at room temperature. The blockingsolution is then removed by aspiration, and the capture plates arefilled with 100 μl/well of stopping reagent (50 μM ATP/20 mM EDTA).

When the three hour enzyme reaction is finished, triplicate 100 μlaliquots of each reaction mix are transferred to three wells containingstopping solution, one well on each of the three capture plates, and thewell contents are mixed well. After one hour at room temperature, thewells of the capture plates are emptied by aspiration and washed fivetimes using PBS and a 12 channel Corning 430474 ELISA plate washer.Finally, 200 μl of Microscint-20 scintillation fluid is added to eachwell of the plate. The plates are coated with plate sealers, then lefton a shaker for 30 minutes. Each capture plate is counted in a PackardTopCount scintillation counter (Meridian, Conn.) and the results areplotted as a function of compound concentration.

Compounds identified using this method can be further optimized bytesting their ability to bind to truncated GSK3 polypeptides of theinvention, using the fluorescence polarization assay, for example, fortruncated polypeptides that do not exhibit GSK3 kinase activity.Alternatively, a truncated GSK3 polypeptide of the invention can be usedin place of the native GSK3 protein.

Example 5 Screening for Inhibition of Tau Protein Phosphorylation

A. Transient Transfection of COS Cells with Expression Plasmid EncodingTruncated GSK3 and Tau Expression Plasmid Construction

COS cells are maintained in T25 tissue culture flasks in high glucoseMEM medium/5% fetal bovine serum. Cells from a confluent T25 flask areharvested and 80,000 cells/well are seeded into Corning 6-well tissueculture plates in a final volume of 2 ml/well of medium. The cells areleft to grow at 37° C. for 48 hours. The cells are then washed twice inOpti-MEM containing no fetal bovine serum, and finally the cells areleft in 1 ml of Opti-MEM.

Polynucleotide encoding tau protein is subcloned into plasmid pSG5 underan early SV40 promoter to generate a tau expression plasmid. The cloningof cDNA encoding tau protein is generally described in Goedert et al.,EMBO Journal, 8(2):393-399 (1989), which is incorporated herein byreference. A GSK3 expression plasmid is prepared by subcloningpolynucleotide encoding truncated GSK3 into pCG, which is an ApEVRFderivative described in Giese et al., Genes & Development, 9:995-1008(1995) and Matthias et al., Nucleic Acid Research, 17:6418 (1989), bothof which are incorporated herein by reference. The polynucleotide canencode any of the truncated GSK3 polypeptides of the invention.

The following solutions are prepared in 1.5 ml Eppendorf tubes:

Solution A: for each transfection, 2 μg of DNA (tau expression plasmid)and 0.7 μg of DNA (GSK3 expression plasmid) are diluted into 100 μl ofOpti-MEM (Gibco BRL); Solution B: for each transfection, 8 μl ofLipofectamine reagent is diluted into 100 μl of Opti-MEM. The twosolutions are combined, mixed gently, and incubated at room temperaturefor 45 minutes to allow DNA-liposome complexes to form. For eachtransfection, 0.8 ml of Opti-MEM is added to the tube containing thecomplexes. The diluted solution is mixed gently and overlaid onto therinsed cells. The cells are incubated with the complexedDNA/Lipofectamine for 6 hours at 37° C. in a CO₂ incubator. Followingincubation, 1 ml of growth medium (high glucose MEM) with 20% FBS isadded to each well and incubated at 37° C. overnight. The medium isreplaced with fresh, complete medium at 18 hours following the start oftransfection, and the cells are left to grow at 37 ° C. for another 48hours.

B. Tau Phosphorylation Inhibition Assay

Two hours before harvesting, 2 μl of GSK3 inhibitor dissolved in DMSO isadded to each well and incubated at 37° C. After 2 hours the medium isremoved and the cells are rapidly frozen on the plates on dry ice andstored at −70° C. Cells are thawed on ice in the presence of 200 μl oflysing buffer (1% Triton® X-100, 20 mM Tris pH 7.5, 137 mM NaCl, 15%glycerol, 25 μg/ml leupeptin, 1 μg ml pepstatin-A, 1 μM PMSF, 21 μg/mlaprotinin, 50 mM NaF, 50 mM β-glycerophosphate, 15 mM sodiumpyrophosphate, 1 mM sodium orthovanadate). The contents of each well arecentrifuged at 14,000 g, 4° C. for 5 minutes and the supernatantstransferred to clean tubes. At this point the lysates may be stored at−20° C.

C. ELISA to Detect Phosphorylated Tau in Cell Lysates

Immulon 4 strips (Dynatech) are coated with monoclonalanti-phosphorylated tau (AT8, Polymedco, Inc.) at 5 μg/ml in PBScontaining Ca++ and Mg++, 100 μl/well. After overnight incubation at 4°C., the strips are washed twice with washing buffer (PBS containing0.05% Tween® 20) and blocked with PBS containing 1% BSA, 5% normal mouseserum and 0.05% Tween® 20 at room temperature for 1 hour. The strips arewashed 5 times with washing buffer. Lysate (100 μl) diluted 1:10 in PBScontaining 1% BSA, 0.1% NaN₃ is added into each well and incubated atroom temperature for 1 hour. After washing, 100 μl of 0.5 μg/mlbiotinylated monoclonal anti-(non-phosphorylated) tau (HT7, Polymedco,Inc.) in PBS-BSA is added into each well. Strips are washed 5 times andHRP-conjugated streptavidin is added, incubated at room temperature for30 minutes and washed extensively with washing buffer. TMB substrate(Pierce) is used for color development and the reaction is stopped byadding an equal volume of 0.8 M sulfuric acid. Strips are read on anELISA plate reader using a 450 nm filter. The concentration of compoundthat inhibits tau phosphorylation to 50% of the maximal level (i.e.,IC₅₀) is determined by fitting a sigmoidal curve to the plotted data.

Those skilled in the art will recognize, or be able to ascertain, usingnot more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such specific embodimentsand equivalents are intended to be encompassed by the following claims.

All patents, published patent applications, and publications citedherein are incorporated by reference as if set forth fully herein.

1-26. (canceled)
 27. A method of identifying an inhibitor of GSK3-β,comprising exposing a GSK3-β molecule to a putative inhibitor, andmeasuring the specific activity of said GSK3-β molecule, wherein areduction in specific enzymatic activity compared to the specificenzymatic activity in the absence of the putative inhibitor indicatesthat said putative inhibitor is an inhibitor of GSK3-β and wherein saidGSK3-β molecule consists of the amino acid sequence of SEQ ID NO:3. 28.The method of claim 27, wherein said specific activity is Tau proteinphosphorylation.
 29. The method of claim 28, wherein Tau phosphorylationis measured by, (a) transfecting a cell line with Tau and with saidGSK3-β polypeptide; and (b) assaying the phosphorylation of Tau using amonoclonal antibody.
 30. The method of claim 29, wherein said cell lineis a COS cell line.
 31. The method of claim 28, wherein said Tauphosphorylation is measured in a cell-free system by ELISA.
 32. A methodof identifying an inhibitor of GSK3-β, comprising exposing a GSK3-βmolecule to a putative inhibitor attached to a fluorophore, andmeasuring the fluorescence polarization, wherein the presence ofpolarization indicates binding of said inhibitor to the binding site ofGSK3-β, and said GSK3-β molecule consists of the amino acid sequence ofSEQ ID NO:3.
 33. The method of claim 32, wherein said fluorophore isattached to staurosporine.